Free Epstein-Barr virus-induced gene 3 functions as an autonomous immunosuppressive cytokine to maintain immune tolerance.

Epstein-Barr virus-induced gene 3(EBI3) is conventionally viewed as a subunit of IL-27 and IL-35, yet the function of uncomplexed "free" EBI3 is unknown. We show that free EBI3 is an autonomous immunosuppressive cytokine that acts via a gp130/WSX-1/STAT3 axis and epigenetic reprogramming. A free-EBI3-specific sandwich ELISA revealed 23.3 ± 2.2 ng/mL in healthy human sera and equivalent levels in mice; no cross-reactivity with IL-27/IL-35 was observed. Serum free EBI3 was reduced in rheumatoid arthritis(RA; 12.4 ± 1.4 ng/mL) and multiple sclerosis (MS; 14.6 ± 1.4 ng/mL; both p< 0.001) and inversely correlated with disease activity(RA-DAS28-ESR r=-0.67; MS-EDSS r=-0.61). Baseline<15 ng/mL predicted higher flare risk(RA HR = 2.8;MS HR = 3.2;p<0.01). Recombinant free EBI3 activated STAT3 exclusively(EC50≈16 nM), suppressed T-cell proliferation (-40%),IFN-y(-45%) and IL-17(-60%),and drove M2 macrophage polarization (+2.7-fold;p<0.001).Epigenetically,it reduced H3K4me3 at Ifng/Il4 promoters, induced Il10/Tgfb hypomethylation(-35%/-28%), and remodeled 1,021 chromatin-accessible STAT3/NF-kB sites. EBI3-/- mice developed spontaneous colitis and severe EAE; daily free EBI3(1 mg/kg) reversed pathology, whereas IL-27/IL-35 did not. Tissue-targeted delivery(liposomes or nanoparticles) outperformed systemic therapy. Ex-vivo free EBI3 normalized RA/MS patient PBMCs(proliferation-50%; IL-17-52%; p< 0.001). Thus, free EBI3 is a distinct cytokine whose gp130/WSX-1 axis constitutes a biomarker and therapeutic target for inflammatory diseases.